1 st Edition. A precious find. ISOLATE Kits. Nucleic Acid Isolation Guide

Transcription

1 A precious find 1 st Edition ISOLATE Kits Nucleic Acid Isolation Guide

2 A great start leads to an excellent finish Bioline s ISOLATE Kits for nucleic acid isolation are perfect to start the analysis of your samples. The kits are designed for fast and efficient isolation of DNA and RNA from a wide range of biological materials, including animal tissue, cultured cells, buccal swabs, fecal material, bacterial cells, plant tissue, PCR products and agarose gels. ISOLATE Kits are based on a proprietary filter membrane spin column technology which selectively binds the nucleic acids. Subsequent to washing steps, highly pure DNA or RNA is eluted in the final step. The isolated products are ready to use in downstream applications such as PCR, real-time PCR, cloning, sequencing, genotyping, etc. Each kit is supplied with a comprehensive Product Manual which contains detailed protocols and additional helpful information. The kit also includes a separate, splash-proof sheet with bench-top protocols for quick reference. DNA Kits es d ig ts c c u zy m o Pcr En pr el g e Tb el g e Ta at i d ss ti Pl an t m l u r at e c l ca Fe e ia l ls el s Bac te r l cca u B ia c sw ab el ed ed r lt u u C ff in l ta i t Para en d o R em b e u ss ti al im An ls d ts ed ti ss u e Product selection table page ISOLATE Plasmid Mini Kit 04 ISOLATE PCR and Gel Kit 06 ISOLATE Fecal DNA Kit 07 ISOLATE Genomic DNA Mini Kit 08 ISOLATE Plant DNA Mini Kit 09 RNA Kits ISOLATE RNA Mini Kit 10 ISOLATE Plant RNA Mini Kit 11 Column-free isolation TRIsure 12 SureClean 13 2 For more information please visit

3 Contents Bioline has a well established and proven track record providing superior quality molecular biology reagents. With the introduction of a range of nucleic acid isolation kits, we aim to make it easier for our customers to obtain high quality, reliable reagents for every step of their research from a single source. The ISOLATE Kits perfectly complement our well known range of downstream reagents such as DNA polymerases, polymerase mixes, real-time PCR kits, as well as our column-free products for RNA (TRIsure) and DNA (SureClean) isolation. With ISOLATE Kits, you can now count on Bioline to provide you with a great start to your research. Bioline ISOLATE Kits provide High yields and high quality Reproducible results 7 kits to cover a wide range of starting materials 14 detailed protocols Bench-top protocols on separate, splash-proof sheets DNA Kits 04 ISOLATE Plasmid Mini Kit 04 ISOLATE PCR and Gel Kit 06 ISOLATE Fecal DNA Kit 07 ISOLATE Genomic DNA Mini Kit 08 ISOLATE Plant DNA Mini Kit 09 RNA Kits 10 ISOLATE RNA Mini Kit 10 ISOLATE Plant RNA Mini Kit 11 Column-free Isolation 12 TRIsure 12 SureClean 13 General Information 14 Technical support 14 Ordering information 14 Associated products 14 3

4 DNA Kits ISOLATE Plasmid Mini Kit Rapid protocol: 15 minutes/5 preps High purity plasmid DNA: typical A 260 /A 280 ratio >1.8 High yields: up to 13μg from 5ml culture Isolated DNA is ready for downstream applications ISOLATE Plasmid Mini Kit 10 Preps BIO ISOLATE Plasmid Mini Kit 50 Preps BIO ISOLATE Plasmid Mini Kit 250 Preps BIO Isolation of high copy plasmid DNA Isolation of low copy plasmid DNA ISOLATE Plasmid Mini Kit is designed for the rapid and efficient isolation of highly pure plasmid DNA from bacterial cultures using proprietary filter membrane spin column technology. The isolation process combines an alkaline lysis of the harvested bacteria, followed by clarification of the lysate and subsequent specific binding of plasmid DNA directly to the filter of a spin column. Contaminants like macromolecular cellular components and salts are efficiently removed during the washing steps. In the final step, plasmid DNA is eluted in a low salt buffer. The kit shows excellent recovery of plasmid DNA even from low culture volumes (Fig. 1) and reproducible results (Fig. 2). Separate protocols are provided for the isolation of high copy and low copy plasmids. In each case, intact, high purity plasmid DNA is isolated within 15 minutes. Typically, up to 13μg and 18μg plasmid DNA is obtained from 5ml and 10ml cultures, respectively. M M Fig. 1 Excellent recovery of plasmid DNA pgem3z plasmid was isolated from decreasing culture volumes of E. coli using ISOLATE Plasmid Mini Kit and run on 1.5% TAE agarose gel. Lane 1: 5ml Lane 2: 3ml Lane 3: 1ml Lane 4: 0.1ml Lane 5: 0.05ml The isolated DNA shows excellent performance in downstream molecular biology applications such as PCR, transformation, cloning, sequencing (Fig. 3), restriction analysis, etc. M Fig. 2 Reproducible results puc19 plasmid was isolated from 5ml E. coli overnight culture and cut with various restriction enzymes before analysis on 1% TAE agarose gel. Each restriction digest was performed in triplicate. Each lane represents an individual miniprep. 4 For more information please visit

5 DNA Kits Isolation of plasmid DNA Bacterial cell culture ml high copy plasmid (5-10ml low copy plasmid) max. speed, 1 min Discard filtrate Add 500µl Wash Buffer AP Add 250µl (500µl) Resuspension Buffer Discard filtrate Vortex Add 700µl Wash Buffer BP Add 250µl (500µl) Lysis Buffer P. Invert tube 5 times Discard filtrate Add 350µl (600µl) Neutralization Buffer. Invert 5-6 times max. speed, 10 min Discard pellet max. speed, 2 min Discard Collection Tube Add µl Elution Buffer. Incubate 1 min 6000 x g, 1 min Transfer supernatant to Spin Column P Isolated plasmid DNA Clip. 1 BQ 20 WL 10 Sequence: AnJe1_T7 Clipped length: 1045 Samples: Quality: 0-9 Left clip: 20 Bases: Right clip: 1064 Average spacing: Avg. qual. in clip.: Average quality >= 10: 81, 20: 150, 30: 926 >= 30 Page: 1 / G C C A A A G G A T T T C C T C T A G A T A A T T T T G T T T A A C T T T A A G A A G G A G A T A T A C A T A T G A A A T A C C T G C T G C C G A C C G C T G C T G C T G G T C T G C T G C T C C T C G C T G C C C A G C C G G C G A T G G C C A T G G A T A T C G G A A T T A A T T C G G A T C C G A A T T C G A G C T C C G T C G A C A A G C T T G C G G C C G C A C T C G A G C A C C A C C A C C A C C A C C A C T G A G A T C C G G C T G C T A A C A A A G C C C G A A A G G A A G C T G A G T T G G C T G C T G C C A C C G C T G A G C A A T A A C T A G C A T A A C C C C T T G G G G C C T C T A A A C G G G T C T T G A G G G G T T T T T T G C T G A A A G G A G G A A C T A T A T C C G G A T T G G C G A A T G G G A C G C G C C C T G T A G C G G C G C A T T A A G C G C G G C G G G T G T G G T G G T T A C G C G C A G C G T G A C C G C T A C A C T T G C C A G C G C C C T A G C G C C C G C T C C T T T C G C T T T C T T C C C T T C C T T T C T C G C C A C G T T C G C C G G C T T T C C C C G T C A A G C T C T A Fig. 3 Excellent performance of isolated plasmid DNA in downstream applications pet19 plasmid was transformed in DH5 alpha E. coli cells. A single colony was picked and grown overnight. Plasmid was isolated from 3ml culture using ISOLATE Plasmid Mini Kit. Sequencing was carried out based on standard T7 promoter and terminator primers. 5

6 DNA Kits ISOLATE PCR and Gel Kit Product PACK SIZE Cat No. 10 Preps BIO ISOLATE PCR and Gel Kit ISOLATE PCR and Gel Kit 50 Preps BIO ISOLATE PCR and Gel Kit 250 Preps BIO minute protocol for purification of PCR products 15-minute protocol for DNA isolation from gels Excellent recovery rate Isolated DNA is ready for downstream applications Isolation of PCR products Purification of PCR products Isolation of DNA from TAE and TBE agarose gels Purification of DNA from contaminats (enzymes, dntps, etc) Add 500µl Binding Buffer A to Spin Column A placed in a Collection Tube ISOLATE PCR and Gel Kit is designed for the purification of PCR products (Fig. 1), and for the isolation of DNA fragments from TAE and TBE agarose gel slices (Fig. 2). A fast and easyto-follow protocol is given for each application. PCR products are purified in 3 minutes using simple binding and elution steps. Concentrated PCR products ranging between 60bp and 30Kb can be eluted in 10µl buffer with a recovery rate of 75-95%. DNA fragments between 100bp and 30Kb can be extracted from agarose gel slices with an excellent recovery rate of 75-90%. Discard filtrate Place Spin Column A in an Elution Tube Add 50µl PCR mixture Add 10-20µl Elution Buffer Incubate at RT for 1 min Mix by pipetting 6000 x g, 1 min Isolated PCR products The isolated DNA is suitable for downstream applications such as transformation, cloning, sequencing, restriction analysis, etc. DNA extraction from agarose gel Excise DNA fragment with scalpel (max. 300mg) Fig. 1 Purification of PCR products PCR was performed to amplify 500bp, 1.2Kb and 5Kb fragments. The products were purified with ISOLATE PCR and Gel Kit and run on a 1.5% TAE agarose gel. The gel shows complete cleanup of primerdimers. Lanes 1, 3, 5: PCR products before cleanup Lanes 2, 4, 6: PCR products after cleanup using ISOLATE PCR and Gel Kit M M Discard filtrate Transfer to 1.5ml or 2.0ml tube Add 700µl Wash Buffer A Discard filtrate Add 650µl Gel Solubilizer. Incubate at 50ºC for 10 min until gel slice dissolves Repeat wash step max. speed, 2 min Discard filtrate M M Fig. 2 Isolation of DNA from agarose gel Various sized DNA fragments were run on 1% TAE agarose gel and extracted using ISOLATE PCR and Gel Kit. The isolated fragments were again run on 1% TAE agarose gel along with the original fragments. 6 M M Add 50µl Binding Optimizer Place Spin Column A in Elution Tube. Add 30-50µl Elution Buffer. Incubate RT for 1 min Vortex Transfer 750µl to Spin Column A placed in a Collection Tube Isolated DNA M2 M1: HyperLadder I M2: HyperLadder IV 1: Not extracted 2: ISOLATE PCR and Gel Kit 3: Competitor A 4: Competitor B For more information please visit

7 DNA Kits ISOLATE Fecal DNA Kit 15-minute isolation protocol High quality, inhibitor-free DNA No need for organic denaturants or proteinases Non-invasive ISOLATE Fecal DNA Kit 25 Preps BIO ISOLATE Fecal DNA Kit 100 Preps BIO Fecal PCR Kit 25 Reactions BIO Fecal PCR Kit 100 Reactions BIO Isolation of fecal DNA from: Humans Birds Rats, mice Rabbits Cattle Isolation of fecal DNA Vortex Bashing Beads Lysis Tube with fecal sample The use of fecal material can be advantageous as it is non-invasive and large amounts can be collected. The isolation of DNA from feces can be challenging. ISOLATE Fecal DNA Kit is specifically developed for the simple, rapid isolation of high quality DNA from a variety of fecal samples including humans, birds, rats, mice, rabbits, cattle, etc. Bacterial, protist, as well as host DNA can be effectively isolated from 150mg sample of mammalian feces. The procedure is easy and can be completed in as little as 15 minutes. Fecal samples are added directly to a Bashing Beads Lysis Tube and rapidly lysed by bead beating in a vortex, without the use of organic denaturants or proteinases. The DNA is then bound, isolated and purified using spin columns. The eluted DNA, free of contaminants and inhibitors, is ideal for downstream applications such as PCR, microarrays, sequencing, genotyping, etc. Fecal PCR Kit Bioline also provides a separate Fecal PCR Kit which contains all the necessary reagents to isolate DNA from fecal samples and subsequently perform PCR (Fig. 1). In addition to the contents of the ISOLATE Fecal DNA Kit, the Fecal PCR Kit includes ImmoMix, which contains IMMOLASE, a hot-start polymerase premixed with ultra-pure dntps and Mg 2+. The kit has been validated on fecal samples of mouse, rabbit, rat and human origin. Filter lysate with Spin Filter (orange cap) 7000 x g, 1 min Bind, Wash, Elute DNA with Spin Column Filter DNA with Spin Filter (green cap) 8000 x g, 1 min Isolated DNA Isolated DNA M tail 400bp tail 1Kb NTC 400bp NTC 1Kb M tail NTC Fig. 1a PCR amplification from mouse fecal DNA DNA was extracted from various amounts of mouse fecal matter using ISOLATE Fecal DNA Kit. PCR was performed using ImmoMix (BIO-25020) on 2µl and 5µl DNA extract. 400bp and 1Kb fragments of the rn18s gene were amplified and analyzed on 1.5% TAE agarose gel. 1: 6mg 2: 12.5mg 3: 29mg 4: 53.6mg 5: 104mg 6: 139mg Fig. 1b PCR amplification from mouse fecal DNA DNA was extracted from various amount of mouse fecal matter and amplified from 2µl extract as described in Fig. 1a to obtain a 1.8Kb product from the rn18s gene. 7

8 DNA Kits ISOLATE Genomic DNA Mini Kit Rapid protocol: 15 minutes after lysis High purity DNA: typical A 260 /A 280 ratio > 1.7 High yields: up to 100μg genomic DNA Isolated DNA is ready for downstream applications Isolation of genomic DNA from: Animal tissue (up to 50mg) Rodent tail (up to 1cm) Paraffin embedded tissue (up to 25mg) Buccal swabs Eukaryotic cells (up to 5 x 10 6 cells) ISOLATE Genomic DNA Mini Kit 10 Preps BIO ISOLATE Genomic DNA Mini Kit 50 Preps BIO ISOLATE Genomic DNA Mini Kit 250 Preps BIO ISOLATE Genomic DNA Mini Kit Protocol Lyse and Homogenize sample with 400µl Lysis Buffer D +25µl Proteinase K. Vortex for 5 sec. Incubate at 50ºC until sample has lysed ISOLATE Genomic DNA Mini Kit is designed for the rapid and efficient isolation of highly pure genomic DNA from a variety of starting materials such as animal tissue, paraffin embedded tissue, mouse or rodent tail, buccal swabs and eukaryotic cells (Fig. 1). Four optimized protocols are provided for different starting materials. The kit generates reproducible results with every sample (Fig. 2). ISOLATE Genomic DNA Mini Kit uses a proprietary filter membrane spin column technology. The isolation process combines fast lysis of the starting material with Proteinase K, followed by specific binding of DNA directly to the filter in a spin column. Subsequent to washing steps, high quality DNA is eluted with an elution buffer. The isolated DNA is suitable for downstream molecular biology applications such as PCR, transformation, cloning, sequencing, restriction analysis, etc. Add 200µl Binding Buffer D Vortex for 15 sec. Transfer to Spin Column D placed in Collection Tube Discard Collection Tube. Wash with 700µl Wash Buffer D max. speed, 2 min Discard Collection Tube. Place Spin Column D in Elution Tube. Add 200µl Elution Buffer. Incubate at room temperature for 1 min 6000 x g, 1 min Isolated DNA M M Fig. 1 Excellent results from a variety of samples Genomic DNA was isolated from different sample types using ISOLATE Genomic DNA Mini Kit and amplified by PCR using MangoMix (BIO-25033). The products were run on 1.5% TAE agarose gel. Lanes 1 and 6: 18s RNA gene PCR from 3T3 cell genomic DNA Lanes 2 and 7: Beta-Rhodopsin gene PCR from HeLa cell genomic DNA Lanes 3 and 8: 18s RNA gene PCR from mouse lung tissue Lanes 4 and 9: 18s RNA gene PCR from mouse tail clipping Lanes 5 and 10: 16s RNA gene PCR from E. coli M Fig. 2 Reproducible results Mouse genomic DNA was isolated using ISOLATE Genomic DNA Mini Kit and kits from competitors A and B. PCR using MangoMix (BIO-25033) was subsequently performed to amplify a 1.4Kb fragment of the 18s rrna gene. Lanes 1-3: PCR from DNA isolated using ISOLATE Genomic DNA Mini Kit Lanes 4-6: PCR from DNA isolated using Mini Kit from Competitor A Lanes 7-9: PCR from DNA isolated using Mini Kit from Competitor B 8 For more information please visit

9 DNA Kits ISOLATE Plant DNA Mini Kit DNA isolated in 30 minutes High purity DNA: typical A 260 /A 280 > 1.7 Clear, easy to follow instructions Isolated DNA is ready for downstream applications Isolation of genomic DNA from: Fresh plant tissue Frozen plant tissue Lyophilized plant tissue Herbarium specimens ISOLATE Plant DNA Mini Kit is designed for the rapid purification of genomic DNA from a variety of wet or dry plant material, including leaves, bark, roots, fruits, etc. Up to 180mg wet plant material and up to 100mg dry plant material can be processed per spin column. The protocol does not require the use of Proteinase K, which means that all components can be conveniently stored at room temperature. ISOLATE Plant DNA Mini Kit 10 Preps BIO ISOLATE Plant DNA Mini Kit 50 Preps BIO ISOLATE Plant DNA Mini Kit 250 Preps BIO Isolate Plant DNA Mini Kit Protocol Homogenize mg plant tissue under liquid N 2 Add 400µl Lysis Buffer PD + 3µl RNase A. Vortex for 10 sec. Incubate at 65ºC until sample has lysed Add 0.5 volume Binding Buffer PD Vortex vigorously. Transfer to Spin Column PD2 placed in a Collection Tube Discard Collection Tube Wash with 700µl Wash Buffer PD twice ISOLATE Plant DNA Mini Kit shows excellent recovery of plant DNA when different homogenization techniques are used (Fig. 1). High yields are obtained with every miniprep (Fig. 2). The isolated DNA is ready for use in downstream applications such as PCR, real-time PCR, cloning, genotyping, etc. Fig. 1 Excellent recovery of plant DNA with different homogenization techniques 20mg freeze-dried budding leaves of Arabidopsis thaliana were homogenized using liquid nitrogen and with a rotor stator homogenizer. Genomic DNA was isolated using ISOLATE Plant DNA Mini Kit. A 1.4Kb fragment of allene oxide synthase gene was amplified from the isolated DNA using MangoMix (BIO-25033). Lanes 1 and 3: liquid nitrogen ground material Lanes 2 and 4: rotor stator homogenized material Add 100µl Precipitation Buffer. Incubate on ice for 5 min max. speed, 5 min Transfer supernatant to Spin Column PD1 Discard Spin Column PD1 max. speed, 2 min Discard Collection Tube Place Spin Column PD2 in Elution Tube. Add 200µl Elution Buffer. Incubate at RT for 1 min 6000 x g, 1 min Isolated DNA M Fig. 2 High yields of plant genomic DNA Genomic DNA was isolated from 20mg freezedried budding leaves of Arabidopsis thaliana using ISOLATE Plant DNA Mini Kit. Using a 2-fold dilution of the miniprep, a 1.4Kb fragment of allene oxide synthase gene was amplified from the isolated DNA with MangoMix (BIO-25033). Lane 1: 100ng plant genomic DNA Lane 2: 50ng plant genomic DNA Lane 3: 25ng plant genomic DNA Lane 4: 12.5ng plant genomic DNA Lane 5: 6.25ng plant genomic DNA M M 9

10 RNA Kits ISOLATE RNA Mini Kit Rapid protocol: minutes High purity RNA Complete removal of genomic DNA Isolated RNA is ready for downstream applications ISOLATE RNA Mini Kit 10 Preps BIO ISOLATE RNA Mini Kit 50 Preps BIO ISOLATE RNA Mini Kit 250 Preps BIO Isolation of RNA from: Animal tissue Eukaryotic cells Bacterial cells ISOLATE RNA Mini Kit Protocol Homogenize and lyse sample with 450µl Lysis Buffer R Discard filtrate ISOLATE RNA Mini Kit is specially designed for the fast and efficient isolation of extremely pure total RNA from a variety of samples. The kit is compatible with animal tissues, cultured cells and bacterial cells. max. speed, 1 min Discard filtrate Wash with 500µl Wash Buffer AR and 700µl Wash Buffer BR The protocol is easy to follow on a step-by-step basis. The cells are lysed with an optimized lysis buffer, which simultaneously inactivates RNases thus protecting the released RNA. The lysate is then applied to a spin column to selectively remove genomic DNA. There is no need to perform a separate DNase digestion step. The RNA is then bound to a silica membrane. Subsequent wash steps remove the remaining cell debris. Pure, high quality RNA is eluted in the final step with RNase-free water (Fig. 1). The isolated RNA shows excellent performance in downstream applications such as reverse transcription, real-time PCR (Fig. 2), Northern blot analysis, microarrays and RNA protection assays. Transfer supernant to Spin Column R1 Discard Spin Column R1 SAVE FILTRATE Add 1 vol. 70% ethanol to filtrate. Transfer to Spin Column R2 in new Collection Tube. Discard filtrate Place Spin Column R2 in Elution Tube Add 30-80µl RNase-free water. Incubate 1 min x g, 1 min Isolated RNA Fig. 1 High quality RNA RNA was isolated from HeLa cells using ISOLATE RNA Mini Kit and analyzed by the Bioanalyzer 2100 (Agilent Technologies). The quality of RNA was found to be exceptional (RIN: 9.2). Fig. 2 Superior performance in real time applications RNA was isolated from mouse 3T3 cells diluted 10-fold using ISOLATE RNA Mini Kit and Competitor Q s kit. Subsequently, real-time reverse transcriptase reactions were performed using SensiMix SYBR One-Step Kit (QT245). Red traces: RNA isolated using ISOLATE RNA Mini Kit. Green traces: RNA isolated using Competitor Q s kit 1: cells, 2: 1400 cells, 3: 140 cells, 4: 14 cells, 5: 1.4 cells 10 For more information please visit

11 RNA Kits ISOLATE Plant RNA Mini Kit Rapid protocol: 30 minutes after homogenization High purity RNA Complete removal of genomic DNA Isolated RNA is ready for downstream applications Isolation of RNA from: Fresh plant tissue Frozen plant tissue ISOLATE Plant RNA Mini Kit is specially designed for the fast and efficient isolation of extremely pure total RNA from a variety of plant tissues, including leaves, bark, roots, fruits, etc. Up to 100mg starting material can be processed per spin column. ISOLATE Plant RNA Mini Kit 10 Preps BIO ISOLATE Plant RNA Mini Kit 50 Preps BIO ISOLATE Plant RNA Mini Kit 250 Preps BIO Isolate Plant RNA Mini Kit Protocol Homogenize and lyse tissue with 450µl Lysis Buffer APR or BPR. Transfer to 1.5ml tube. max. speed, 1 min Discard pellet Discard filtrate Wash with 500µl Wash Buffer APR and 650µl Wash Buffer BPR The protocol is easy to follow on a step-by-step basis. Two lysis buffers are provided to ensure lysis of different sample types. The buffers also inactivate RNases, thus protecting the released RNA. The lysate is applied to a spin column to selectively remove genomic DNA, eliminating the need to perform a separate DNase digestion step. The RNA is then bound to a silica membrane. Subsequent wash steps remove the remaining cell debris and pure RNA is eluted in the final step with RNase-free water. The isolated RNA shows excellent performance in downstream applications such as real-time PCR (Fig. 1), reverse transcription (Fig. 2), Northern blot analysis, microarrays and RNA protection assays. Transfer supernant to Spin Column PR1 Discard Spin Column PR1 SAVE FILTRATE Add 1 vol. 70% ethanol to filtrate. Transfer to Spin Column PR2 in new Collection Tube. Discard filtrate Place Spin Column PR2 in Elution Tube Add 30-80µl RNasefree water. Incubate 1 min. Isolated plant RNA Fig. 1 Superior performance in real time applications RNA was isolated from 20mg freeze-dried budding leaves of Arabidopsis thaliana using ISOLATE Plant RNA Mini Kit. cdna was synthesized using cdna Synthesis Kit (BIO-65026) and diluted serially 10-fold. Real-time PCR was performed using SensiMix SYBR (QT605) and primers designed against the UBQ10 gene. M M Fig. 2 High yield cdna obtained from isolated RNA RNA was isolated from 20mg freezedried budding leaves of Arabidopsis thaliana using ISOLATE Plant RNA Mini Kit. cdna was synthesized using cdna Synthesis Kit and diluted serially. PCR was performed using MangoMix (BIO-25033) to amplify a 1.4Kb fragment of the allene oxide synthase gene. Products were run on a 1.5% agarose gel. Lane 1: 1µl cdna Lane 2: 2-fold dilution Lane 3: 4-fold dilution Lane 4: 8-fold dilution Lane 5: 16-fold dilution Lane 6: 32-fold dilution 11

12 Column-free Isolation TRIsure Isolation of high-quality RNA in 60 minutes Ready-to-use solution for a wide variety cells and tissues Cost-effective and simple protocol Isolated RNA is ready for downstream applications TRIsure 100ml BIO TRIsure 200ml BIO TRIsure Plus Bacterial RNA Isolation Kit 100 Preps BIO TRIsure Plus Bacterial RNA Isolation Kit 200 Preps BIO Bacterial Enhancement Reagent 20ml BIO Isolation of RNA from: Animal tissues Cultured cells Plant tissues Bacterial cells TRIsure is a ready-to-use reagent for the isolation of high quality total RNA from diverse biological materials, including animal tissues and cells, as well as plant tissues rich in polysaccharides and proteoglycans. TRIsure maintains the integrity of the extracted RNA, while disrupting cells and subsequently dissolving cell components. The biological sample is homogenized or lysed in TRIsure and then separated into organic and aqueous phases. The RNA remains in the aqueous phase and is subsequently recovered by precipitation with isopropyl alcohol. High yield (table 1) and high quality RNA is extracted from various samples (Fig. 1). The isolated RNA is suitable for any downstream application such as RT-PCR, hybridization assays, and in vitro translation. 1ml of TRIsure is sufficient to isolate total RNA from 1 x 10 7 cells or 100mg of tissue. TRIsure Plus Bacterial RNA Isolation Kit To enhance the isolation of RNA from bacterial cells, we recommend the use of TRIsure Plus Bacterial RNA Isolation Kit. The kit contains a specially developed proprietary Bacterial Enhancement Reagent, in addition to TRIsure. A simple pre-treatment of the sample with the Bacterial Enhancement Reagent, prior to using TRIsure, facilitates the isolation of high purity, intact RNA by degrading proteins and inactivating endogenous RNases (Fig. 2). Higher yields (table 2) are obtained due to improved lysis of bacterial cells, eliminating the need for time consuming mechanical or enzymatic lysis steps. Fig. 1 High quality and yield of RNA extracted using TRIsure. RNA extracted from 3T6 cells and mouse tissue, using TRIsure and Competitor reagent. Lane 1: 4µg of total RNA from 3T6 cells Lane 2: 4µg of total RNA from mouse kidney tissue Lane 3: 4µg of total RNA from mouse liver tissue M: RiboLadder Long Table 1. Expected yield of RNA from different samples using TRIsure Sample type Sample quantity Expected yield Cultured epithelial cells 1 x µg Cultured fibroblasts 1 x µg Mouse kidney tissue 1mg 2-5µg Mouse liver tissue 1mg 5-10µg Table 2. Expected yield of RNA from different bacteria using TRIsure Plus Bacterial RNA Isolation Kit Sample type Sample quantity Expected yield Gram-negative bacteria (e.g. E. coli) 1 x 10 8 >30µg Gram-positive bacteria (e.g. Lactococcus lactis) 1 x 10 8 ~3µg Bacterial Enhancement Reagent is also available as a separate product (BIO-38037). Fig. 2 Isolation of high quality RNA 1.5ml log phase culture of Bacillus subtilis was pre-treated with Bacterial Enhancement Reagent, followed by isolation of RNA using TRIsure. The RNA was analyzed using the Bioanalyzer 2100 (Agilent Technologies) and was found to be of high quality and purity. 12 For more information please visit

13 Column-free Isolation SureClean Column-free PCR clean-up Post-PCR recovery up to 98% Cost-effective and simple protocol Isolated products are suitable for downstream applications SureClean 5ml BIO SureClean 25ml BIO SureClean Plus 5ml BIO SureClean Plus 25ml BIO Removes primers, non-specifics, dntps and restriction enzymes DNA or dsrna purification or concentration Buffer exchange SureClean Protocol SureClean is a novel, inexpensive solution, which provides a column-free method for nucleic acid purification. Using a simple and rapid procedure, SureClean can be used to purify or concentrate DNA or dsrna from PCR reactions or any enzymatic digests. This method is easy to follow, combining convenience, speed and excellent recovery rates. A very straightforward protocol allows the precipitation of nucleic acids 75bp with up to 98% recovery of the original sample without the need for organic solvents, glass milk or expensive spin columns (Fig. 1). SureClear enable the researcher to resuspend the cleaned-up nucleic acids in any buffer and volume of choice, thus permitting the purification process to be tailored specifically to suit the experiment. SureClean purifies nucleic acids without the use of chaotropic salts (which often contribute to denaturation of the DNA duplex). SureClean is compatible with downstream applications, such as cloning and sequencing. Optional step (SureClean Plus only): Add pink Co-Precipitant Add an equal volume of SureClean to the nucleic acid sample Incubate at room temperature for 10 minutes Centrifuge SureClean Plus A version of SureClean, known as SureClean Plus, incorporates a pink co-precipitant that facilitates easy visualization of the purified pellet. The co-precipitant does not interfere and so is suitable for use in standard PCR, real-time PCR and other enzymic reactions. Aspirate supernatant 70% Ethanol wash Resuspend pellet in desired buffer 200bp 75bp 50bp 25bp DNA purification using SureClean and Competitor Q s & X s spin column purification methods. 30µl of HyperLadder V was purified using the manufacturer s protocols. For each of the methods DNA was resuspended in 30µl TE, of which 5µl was loaded on to a 3.5% agarose gel. Lane 1: HyperLadder V Lane 2: HyperLadder V purified using SureClean Lane 3: HyperLadder V purified using spin-columns from Competitor X Lane 4: HyperLadder V purified using spin-colums from Competitor Q 13

14 General Information Technical SUPPORT For technical assistance or more information on these products, please call us on: UK: +44 (0) US: DE: +49 (0) AUST: +61 (0) INT: +44 (0) or us at ORDERING INFORMATION ASSOCIATED PRODUCTS ISOLATE Plasmid Mini Kit 10 Preps BIO ISOLATE Plasmid Mini Kit 50 Preps BIO ISOLATE Plasmid Mini Kit 250 Preps BIO ISOLATE PCR and Gel Kit 10 Preps BIO ISOLATE PCR and Gel Kit 50 Preps BIO ISOLATE PCR and Gel Kit 250 Preps BIO ISOLATE Fecal DNA Kit 25 Preps BIO ISOLATE Fecal DNA Kit 100 Preps BIO Fecal PCR Kit 25 Reactions BIO Fecal PCR Kit 100 Reactions BIO ISOLATE Genomic DNA Mini Kit 10 Preps BIO ISOLATE Genomic DNA Mini Kit 50 Preps BIO ISOLATE Genomic DNA Mini Kit 250 Preps BIO ISOLATE Plant DNA Mini Kit 10 Preps BIO ISOLATE Plant DNA Mini Kit 50 Preps BIO ISOLATE Plant DNA Mini Kit 250 Preps BIO ISOLATE RNA Mini Kit 10 Preps BIO ISOLATE RNA Mini Kit 50 Preps BIO ISOLATE RNA Mini Kit 250 Preps BIO ISOLATE Plant RNA Mini Kit 10 Preps BIO ISOLATE Plant RNA Mini Kit 50 Preps BIO ISOLATE Plant RNA Mini Kit 250 Preps BIO TRIsure 100ml BIO TRIsure 200ml BIO TRIsure Plus Bacterial RNA Isolation Kit 100 Preps BIO TRIsure Plus Bacterial RNA Isolation Kit 200 Preps BIO Bacterial Enhancement Reagent 20ml BIO SureClean 5ml BIO SureClean 25ml BIO SureClean Plus 5ml BIO SureClean Plus 25ml BIO Agarose, Molecular Grade 500g BIO MangoMix 250 Reactions BIO HyperLadder I 500 Lanes BIO HyperLadder IV 500 Lanes BIO IMMOLASE DNA Polymerase 500 Units BIO ImmoMix 500 Reactions BIO SensiMix SYBR One-Step Kit 250 Reactions QT cdna Synthesis Kit 100 Reactions BIO A precious find ISOLATE Kits Nucleic Acid Isolation Guide To download the electronic version of this guide, please go to 1 st Edition TRIsure, HyperLadder, IMMOLASE, ImmoMix, MangoMix are trademarks of Bioline. SensiMix is a trademark of Bioline Reagents Ltd. 14 For more information please visit

15 Laboratory workflow Products available at Bioline Sample Animal tissue Cultured cells Fecal material Bacterial cells Plant tissue Nucleic Acid Purification DNA/RNA ISOLATE Kits TRIsure SureClean Proteinase K Amplification PCR DNA polymerases Polymerase mixes dntps Analysis Real-time PCR SYBR-based kits Probe-based kits Control templates Gel electrophoresis Agarose Electrophoresis buffers Ladders Downstream Processing Cloning Competent cells Ligases Antibiotic solutions Cloning reagents Reverse transcription One-step RT-PCR kit cdna Synthesis cdna synthesis kits

16 United Kingdom Bioline Ltd 16 The Edge Business Centre Humber Road London NW2 6EW Tel: +44 (0) Fax: +44 (0) Germany Bioline GmbH Im Biotechnologiepark, TGZ 2 D Luckenwalde Tel: +49 (0) Fax: +49 (0) info.de@bioline.com Australia Bioline (Aust) Pty Ltd PO Box 122 Alexandria NSW 1435 Tel: +61 (0) Fax: +61 (0) info.aust@bioline.com USA Bioline USA Inc. 305 Constitution Drive Taunton MA Tel: Fax: Order Toll Free: info.us@bioline.com PG0910V2.2